JPH0543350B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an additive used in a cell culture medium in cell culture technology, and specifically relates to a new cell culture additive that can set favorable cell culture conditions.

BACKGROUND OF THE INVENTION With recent advances in biotechnology, cell culture technology as a basic technology has become increasingly important, and much research has been carried out particularly on media suitable for cell culture.

Currently, various types of such cell culture media (nutrient media, basal media) are commercially available, but as is generally known, these media require the addition of fetal calf serum (FCS). However, fetal bovine serum has the drawbacks of being difficult and expensive to obtain, and having uneven effects on culture. In addition, when useful substances are produced and collected by cell culture using a medium supplemented with fetal bovine serum, the culture solution contains an extremely wide variety of components, including unknown components derived from the fetal bovine serum. However, it may be very difficult to separate this from the target useful substance, and sometimes it may even be impossible to isolate the target substance.

On the other hand, bovine serum (excluding fetal serum, the same applies hereinafter)
Although it is easily available at low cost and in large quantities, it does not have the effect of supporting cell proliferation as seen in fetal bovine serum.

Problems to be Solved by the Invention The present invention has a cell proliferation effect as described above,
A new cell culture additive that can be used in place of fetal bovine serum, which must be added to cell culture media but has various drawbacks, and which does not have the drawbacks of fetal bovine serum. The purpose is to provide a drug.

Means for Solving the Problems The above purpose is to obtain a treated supernatant obtained by heat-treating bovine serum at a temperature of 80 to 100℃ (hereinafter referred to as
sup"), and contains at least one fraction that elutes at pH 4.5, 5.0, and 6.0 (0.1M acetic acid-sodium acetate buffer) in ion exchange chromatography using a carboxymethylcellulose column. This is achieved by a cell culture additive characterized by the following.

The active ingredient of the cell culture additive of the present invention has the effect of maintaining cell functions and promoting cell proliferation extremely well. In addition, since unnecessary components are precipitated and removed through heat treatment, when added to the culture medium of cells that produce useful substances, contamination with impurities is prevented and useful substances are isolated. It has the advantage of being easier to separate, and the problem of microbial contamination can also be avoided. Furthermore, the active ingredient has the characteristic that it has a uniform effect on culture, and for these reasons, it can be used particularly effectively when testing the cytotoxicity of various drugs using cultured cells. can.

In the cell culture additive of the present invention, the above BS
-sup and pH set in 0.1M acetic acid-sodium acetate buffer in ion exchange chromatography using a carboxymethyl cellulose column.
Fractions characterized by eluting at 4.5, 5.0, and 6.0 (hereinafter referred to as "F-1" and "F-1", respectively)
-2" and "F-3") may be contained alone or in combination of two or more thereof as an active ingredient, and furthermore, the above BS-sup containing all of these active ingredients may be used as is. You can also do it.

The cell culture additive of the present invention will be described in detail below, starting with its manufacturing method.

As the bovine serum used in the production of the additive of the present invention, various commercially available products can be used.
It is also possible to use blood collected from cows or blood obtained at a slaughterhouse, separated and purified in a conventional manner.

The above heat treatment of bovine serum can be carried out by diluting the bovine serum itself or, if necessary, diluting it with an appropriate aqueous solvent, such as a normal buffer such as physiological saline or phosphate buffer, at a temperature of 80 to 100%. ℃, preferably about 85 ~
This is carried out by heating at 90°C for about 20 to 30 minutes.

According to the above heat treatment, a precipitate is deposited, and by removing this precipitate, the BS-sup of the present invention is obtained. Removal of this precipitate can be carried out according to conventional methods, such as filtration or centrifugation.

In addition, F-1 and F as purified products of the above BS-sup
-2 and F-3 are obtained by appropriately treating BS-sup according to general physical and biochemical operations such as ion exchange chromatography using a normal ion exchanger and molecular sieve chromatography. and identified as the above fraction.

Thus BS-sup, F-1, F-2 and F-3
are obtained, and these can be further subjected to conventional purification means such as dialysis, freeze-drying, etc., as necessary.

The cell culture additive of the present invention contains the active ingredients (BS-sup, F-1, F-2) obtained as described above.
As long as it effectively contains F-3), there is no particular restriction on its form, for example, it can be advantageously used as the cell culture additive of the present invention in the form of an aqueous solvent solution containing only the active ingredient. can.

Further, the additive of the present invention can be blended with a usual diluent, a component of a nutrient medium, a growth supporting component, a preservative, etc., and the present invention provides a form in which additives such as such a diluent are blended. Naturally, it also includes those of

The additive of the present invention can be used for cell culture by adding it to a general basal medium for cell culture. The above-mentioned basal medium is not particularly limited, and any known variety can be used. As a specific example,
For example, RITC medium, CEM medium, CMRL-1066 medium, DM-160 medium, Eagle's minimum essential medium (Eagle's MEM), autoclavable MEM,
Fisher's Medium, F-10
Examples of the medium include F-12 medium, L-15 medium, NCTC-109 medium, and RPMI-1640 medium.

The amount of the additive of the present invention to be added to the above medium can be arbitrarily determined and is not particularly limited, but the amount of the additive of the present invention added to the above medium can be determined arbitrarily.
BS-sup or F-1, F as an active ingredient for
The total protein amount of at least one of -2 and F-3 is about 0.1 μg to 100 μg, preferably about 1 to 30 μg.

The method of culturing cells using the cell culture medium to which the additive of the present invention has been added is not particularly different from conventional cell culture methods, and requires appropriate pH and temperature conditions suitable for the growth of the cells to be cultured. Lower, normal PH
The culture may be carried out at a temperature of about 6 to 8°C and a temperature of about 37°C.

Cells that can be cultured using a medium supplemented with the cell culture additive of the present invention are not particularly limited, but cells of animal origin are preferred. The cells may be adherent cells or floating cells, or may be already established cell lines, fresh malignant tumor cells, or normal cells, and any of these cells may be cultured in a medium supplemented with the additive of the present invention. Good proliferation is observed.

Examples Examples and test examples are given below to explain the present invention in more detail.

Example 1 Phosphate buffered saline (PBS, PH=7.4)
100 ml of bovine serum diluted 2 times at 90℃ in a water bath.
Heat for 30 minutes. After heating, the reaction solution was centrifuged (5000 rpm x 30 minutes) to obtain a supernatant. After dialyzing this supernatant against PBS overnight, 10 mg protein/
ml. This is called "Additive No. 1".

The supernatant obtained above was applied to a CM-52 column [Whatman; diameter 1.6 x 40 cm, eluent: 0.1 M acetic acid-sodium acetate buffer, 3 ml/tube], and the pH was adjusted to 4.5, 5.0, and 6.0. Obtain the elution fraction of
Each of these was adjusted to have a protein content of 1.0 mg/ml. These are sequentially referred to as "Additive No. 2,""Additive No. 3," and "Additive No. 4."

Test example 1 Human cell line HEL [Human embryonic lung]
fibroblast; by Kuruse and Patterson, Tissue
Culture, p336, Academic Press,
NewYork, 1973 edition] in RITC medium [Cell Str.Func., without bovine serum albumin (BSA)].
1279 (1976)] at 1.0×10 ⁴ cells/dish and cultured in the presence of additive No. 1 at various concentrations. The number of living cells was counted after 6 days. The results are shown in graph (1) in Figure 1. In the figure, the vertical axis represents the number of cells (×10 ⁴ cells/date).
The horizontal axis represents the amount of the test drug (additive) added (μg protein amount/ml), and this amount O represents the control. Figure 1 also shows the results when _α1 acid glycoprotein (AGP) was added as a control drug.
Also listed as (2).

It is clear from FIG. 1 that the use of the additive of the present invention results in extremely significant cell proliferation.

In the above, HeLa-S3 instead of HEL
[Human cervical carcinoma; JEM103273
(1956)], the cell proliferation activity of the additive of the present invention was tested in the same manner. The results are shown in Figure 2, and according to the addition of the additive of the present invention, graph (1)
As shown, it can be seen that the proliferation of the HeLa-S3 cells can be significantly promoted even when compared with the addition of AGP (shown in graph (2)).

In the above, Yoshida sarcoma (YS) was used instead of HEL; Hiroshi Sato, "List of transplantable tumors maintained in Japan (Japanese Cancer Society general meeting article reprint)", p. 51, 1982. The cell proliferation activity of the additive of the present invention was tested in the same manner except that the cells were cultured for 4 days using primary cultured cells (inoculated at 1.0 x 10 ⁴ cells/ml) in October (Japan Cancer Society). . The results are shown as graph (1) in Figure 3, and the AGP is shown as graph (2).
It was confirmed that good cell proliferation activity was achieved which cannot be achieved by using .

In the above, additive No. 1 is replaced with additive No. 1.
2. Inoculum amount of each of Additive No. 3 and Additive No. 4
A similar test was performed using 1.0×10 ⁴ cells/well. The results are shown in Figure 4.

In the figure, graph (3) shows the use of additive No. 2.
Graph (4) shows the use of additive No. 3, and graph (5)
indicate the use of additive No. 4, respectively.

From FIG. 4, it can be seen that each of the above-mentioned additives also has excellent cell proliferation activity similar to Additive No. 1.

[Brief explanation of the drawing]

FIG. 1 to FIG. 4 are graphs showing the results of examining the proliferation properties of various cells when cultured using a medium supplemented with the cell culture additive of the present invention.

Claims (1)

[Claims] 1 The treated supernatant obtained by heat-treating bovine serum at a temperature of 80 to 100°C, which has a pH of 4.5, 5.0, and 6.0 (0.1M acetic acid-sodium acetate), was analyzed by ion exchange chromatography using a carboxymethyl cellulose column. An additive for cell culture, characterized in that it contains at least one fraction eluted in a buffer solution.